Endocrine Abstracts

Peptide YY (PYY) is secreted by enteroendocrine L-cells in response to nutrient and microbiota-derived stimuli. Acting through neuropeptide Y (NPY) receptors, PYY regulates central appetite pathways, modulates gastrointestinal motility, and exerts growth factor–like trophic effects on epithelial and endocrine tissues. NPY receptor agonism via PYY analogs (e.g PYY1875, BI 1820237, NNC0165-1273) and multi-receptor agonists (e.g GEP44, bGLP/PYY-19) is in early stages of investigation for weight loss and glucose-lowering efficacy, exhibiting promising preclinical and clinical outcomes. However, in-vitro screening of PYY secretagogues is limited by a lack of scalable human models: immortalised lines express PYY poorly, while intestinal organoids, though physiologically relevant, require complex differentiation protocols yielding low and variable proportions of enteroendocrine cells. We present a fluorescent PYY biosensor, super-ecliptic phluorin (SEP)-tagged PYY (SEP-PYY), for functional high-throughput and cost-effective readout of PYY synthesis and secretion in real-time, in human enteroendocrine NCI-H716 cells. Iterative construct design of SEP-PYY by computational methods ensured faithful pro-hormone processing of SEP-PYY and targeting to secretory vesicles in NCI-H716 cells which we validated by super-resolution confocal microscopy. SEP-PYY failed to form secretory granules in non-enteroendocrine cells, confirming dependence on endogenous enteroendocrine machinery. We developed protocols for detection of SEP-PYY production by flow cytometry and secretion by spectrofluorometric plate readout. SEP-PYY responded robustly to acute and chronic nutrient stimulation, mirroring endogenous PYY release. Notably, butyrate upregulated SEP-PYY production and secretion in a dose- and time-dependent manner. Finally, we leveraged the pH sensitivity of SEP to capture single-event exocytosis events of SEP-PYY at the plasma membrane in live NCI-H716 cells by total internal reflection microscopy (TIRF). Fusion of acidic granules with the extracellular milieu produced ~1000 ms fluorescence flashes, which were significantly upregulated by butyrate treatment. In summary, our study provides a scalable, real-time platform for investigating nutritional and pharmacological regulation of gut hormone secretion.